MicroRNAs (miRNAs) are short regulatory RNAs of ˜22 nucleotide (nt) that are expressed by all metazoan eukaryotes. For example, humans encode 420 or more distinct miRNAs. miRNAs are ordinarily first transcribed by RNA polymerase II in the form of a long, capped, polyadenylated transcript called a primary miRNA (pri-miRNA). The miRNA forms part of one arm of an ˜80 nt imperfect stem-loop within the pri-miRNA. One pri-miRNA may contain a cluster of several miRNA stem-loops.
The first step in miRNA biogenesis is the recognition of the pri-miRNA hairpin by the nuclear RNase III enzyme Drosha. Drosha cleaves the RNA hairpin approximately two-thirds of the way down the stem, leaving a 2 nt 3′ overhang, to liberate an ˜60 nt RNA hairpin called a pre-miRNA. This pre-miRNA is exported to the cytoplasm where a second RNase III enzyme, Dicer, binds the base of the pre-miRNA and cleaves ˜22 nt away, generating a second 2-nt 3′ overhang. The mature miRNA, which forms one strand of the resulting miRNA duplex intermediate, is then incorporated into the RNA induced silencing complex (RISC) where it acts as a guide RNA to target RISC to complementary mRNAs. Once bound to an mRNA, RISC ordinarily can induce mRNA cleavage and degradation, if the complementarity is extensive, or translation arrest, if the complementarity is partial. Post-transcriptional regulation by miRNAs is now known to represent a major level of gene regulation in eukaryotes and miRNAs have been shown to regulate many aspects of cell differentiation and development.
In addition to cells, it is now known that several viruses, including, herpesviruses, encode and express miRNAs in infected cells. Specifically, the human γ-herpesviruses EBV and KSHV have been shown to express 23 and 12 miRNAs, respectively, while the human β-herpesvirus hCMV expresses 11 miRNAs. Less is known about the human α-herpesviruses herpes simplex virus type 1 (HSV-1) and HSV-2.
Initial infection by HSV-1 generally occurs at mucous membranes, frequently around the mouth and lips, and subsequently in trigeminal ganglion neurons that innervate these tissues, and results in a localized infection that resolves, leaving behind trigeminal ganglia that maintain a latent infection with HSV-1. The virus occasionally spontaneously reactivates, often in response to some form of stress, leading to localized painful “cold sores”. Once established, latent HSV-1 infection is life-long and, currently, cannot be cured, although the severity of outbreaks can be ameliorated with antiviral drugs. In immunocompromised patients, HSV-1 can become a serious infection, leading to neuronal damage or even death.
In the latently infected trigeminal ganglion, the HSV-1 genome is found exclusively in infected neurons. Within these neurons, HSV-1 is thought to express a ˜8.3 kb viral transcript, the so-called minor latency associated (LAT) RNA (Bloom, int. Rev. Immunol. 23:187-198 (2004); Stevens et al, Science 235:1056-1059 (1987)) (FIG. 1). LAT is spliced, to give rise to the abundantly expressed ˜2 kb LAT intron, but the remaining exonic—6.3 kb RNA is highly unstable (FIG. 1).
HSV-2 is a close relative of HSV-1, with ˜80% sequence identity at the genome level, but generally infects via genital mucous membranes to establish latent infections in the sacral ganglia that innervate the genitals. Like HSV-1, HSV-2 establishes latent infections in neurons and expresses a LAT in those cells.
The present invention results, at least in part, from studies designed to test the hypothesis that HSV-1 and/or HSV-2 LAT might be a pri-miRNA, that is, that one function of LAT may be to generate miRNAs that play a key role in viral latency.